P746: COMBINATION OF RAS/MAPK MODULATOR AND AZACITIDINE AFFECTS HISTONE MARKS AND IMPACTS THE INNATE IMMUNE SIGNALING PATHWAY IN THE MDS-L CELL LINE
Authors: R. Rai, F. Patel, J. Feld, S. Melana, S. Navada, R. Odchimar-Reissig, E. Demakos, E. P. Reddy, A. Horowitz, L. Silverman
Journal: HemaSphere (2022)

Background: Myelodysplastic syndrome (MDS) is a stem cell disorder characterized by ineffective hematopoiesis, and a propensity to transform into acute myeloid leukemia. MDS patients exhibit chronic activation of the innate immune response and a hyperinflammatory microenvironment (Barryero L, et al. Blood, 2018). Azacitidine (Aza), as a single agent has an overall response rate (ORR) of only 50% (Silverman LR, et al., JCO 2002; Fenaux P et al., Lancet Oncol. 2009). The response to Aza is not durable and all patients relapse with worsening bone marrow failure. Aza combined with a multi-kinase inhibitor of RAS and MAPK pathways, rigosertib (RAS/MAPK), reverses the bone marrow failure state and demonstrates an ORR of 54% in patients who failed a prior HMA based therapy. (Navada SC, et al. EHA Library 2019). This represents a critical observation in overcoming the epigenetic clinical resistance phenotype, though the mechanism is still elusive. Aberrant RAS/MAPK signaling has also contributed to MDS pathophysiology. Aims: To investigated the impact of RAS modulation combined with Aza on the innate immune signaling pathway. Methods: We used the MDS-L cell line as a model to limit the heterogeneity observed in MDS patients. The cells were treated with, Aza, RAS/MAPK and RAS/MAPK-Aza for 48 hrs and further analyzed by qPCR and western blot. We studied the expression of several histone proteins and various pattern recognition receptors (TLRs, RIG-I, MDA5, STING), their intermediate adaptor molecules myeloid differentiation factor 88 (MYD88), mitochondrial antiviral signaling (MAVS) gene; and interferon regulatory factor (IRF)-3 and -7 by qPCR. Results: We found an increase in H3K9ac protein expression with RAS/MAPK and RAS/MAPK-Aza; Aza and RAS/MAPK alone each had similar effects on H3K4me3, however, its expression was markedly upregulated with RAS/MAPK-Aza. Effects on H3K36me3 were comparable in all treated cells. We observed marked effects on the repression marks H3K9me3 and H3K27me3 by RAS/MAPK and RAS/MAPK-Aza combination. Furthermore, we observed that Aza, RAS/MAPK and RAS/MAPK-Aza significantly inhibit the expression of TLR1, 2 and 6. However, TLR-3, 9, RIG-I, MDA5, STING, MAVS, MYD88, and IRF-3, 7 were significantly inhibited by RAS/MAPK and RAS/MAPK-Aza. Summary/Conclusion: We find that the RAS/MAPK modulator rigosertib has histone modifying effects alone and when combined with Aza. The combination has effects on histone modification of both activator and repressor marks and is associated with down-regulation of the innate immune signaling pathway. In the clinic RAS/MAPK modulation combined with Aza reverses bone marrow failure in MDS. Further studies are underway to determine the correlation of the histone modification and innate immune signaling changes, as well as the role of RAS/MAPK modulation, to determine how these mechanisms contribute to the improvement in hematopoiesis in MDS patients.